Human Hepatitis B virus (HBV) is a DNA virus that causes acute and chronic hepatitis and is associated with the development of primary hepatocellular carcinoma. The study of HBV biology has been hampered by the inability to grow the virus efficiently in tissue culture. Therefore, most information about HBV has been derived from cloning and sequencing the HBV genome and the expression of individual cloned HBV genes in tissue culture. These studies have revealed that the viral DNA contains four open reading frames encoding surface antigens, core antigens, the viral polymerase and a protein termed X due to its unknown function. Only three major classes of viral mRNAs have been reported: a 2.4-kilobase (kb) and a 2.1-kb subgenomic RNA encoding presurface and surface antigens, respectively, and a 3.5-kb pregenomic RNA that can potentially be translated utilizing three open reading frames encoding core antigen, polymerase and X protein. The aim of this proposal is to understand how sequences in the 3.5-kb pregenomic mRNA can encode three proteins. We will test whether RNA sequences located upstream of open reading frames encoding polymerase and X proteins can bind ribosomes internally in human liver cells, allowing the pregenomic mRNA to function as a polycistronic mRNA molecule. To accomplish this, a sensitive assay will be employed, that we have used to demonstrate internal ribosome binding to the 5'noncoding region of both poliovirus RNA and antennapedia mRNA of Drosophila melanogaster. The experimental approach will be to construct hybrid genes, containing first a reporter gene (chloramphenicol acetyltransferase, CAT), followed by an intracistronic spacer (ICS) region and a second, downstream reporter gene (firefly luciferase, LUC). Sequences upstream of the coding region for the polymerase and X gene, respectively, will also be used as ICS sequences. These plasmids will be transcribed in vitro by T7 RNA polymerase producing chimeric, dicistronic RNA molecules containing 5'-CAT- intracistronic spacer-LUC-3' sequences. These RNA molecules will then be transfected into hepatic and nonhepatic cells and the translation of LUC protein will be monitored. Translation of LUC indicates that the intracistronic spacer sequences can bind ribosomes internally. Further experiments will be designed to identify RNA sequences and RNA/protein complexes that participate in pregenomic mRNA translation. Thus, combining genetic and biochemical approaches to study translation of HBV will contribute to the elucidation of events governing the initiation of translation in human liver cells.